Short-term high-fat diet feeding of mice suppresses catecholamine-stimulated Ca2+ signalling in hepatocytes and intact liver.

Excess consumption of carbohydrates, fat and calories leads to non-alcoholic fatty liver disease (NAFLD) and hepatic insulin resistance; these are major factors in the pathogenesis of type II diabetes. Hormones and catecholamines acting through G-protein coupled receptors (GPCRs) linked to phospholipase C (PLC) and increases in cytosolic Ca2+ ([Ca2+ ]c ) regulate many metabolic functions of the liver. In the intact liver, catabolic hormones such as glucagon, catecholamines and vasopressin integrate and synergize to regulate the frequency and extent to which [Ca2+ ]c waves propagate across hepatic lobules to control metabolism. Dysregulation of hepatic Ca2+ homeostasis has been implicated in the development of metabolic disease, but changes in hepatic GPCR-dependent Ca2+ signalling have been largely unexplored in this context. We show that short-term, 1-week, high-fat diet (HFD) feeding of mice attenuates noradrenaline-stimulated Ca2+ signalling, reducing the number of cells responding and suppressing the frequency of [Ca2+ ]c oscillations in both isolated hepatocytes and intact liver. The 1-week HFD feeding paradigm did not change basal Ca2+ homeostasis; endoplasmic reticulum Ca2+ load, store-operated Ca2+ entry and plasma membrane Ca2+ pump activity were unchanged compared to low-fat diet (LFD)-fed controls. However, noradrenaline-induced inositol 1,4,5-trisphosphate production was significantly reduced after HFD feeding, demonstrating an effect of HFD on receptor-stimulated PLC activity. Thus, we have identified a lesion in the PLC signalling pathway induced by short-term HFD feeding, which interferes with hormonal Ca2+ signalling in isolated hepatocytes and the intact liver. These early events may drive adaptive changes in signalling, which lead to pathological consequences in fatty liver disease. KEY POINTS: Non-alcoholic fatty liver disease (NAFLD) is a growing epidemic. In healthy liver, the counteracting effects of catabolic and anabolic hormones regulate metabolism and energy storage as fat. Hormones and catecholamines promote catabolic metabolism via increases in cytosolic Ca2+ ([Ca2+ ]c ). We show that 1 week high-fat diet (HFD) feeding of mice attenuated the Ca2+ signals induced by physiological concentrations of noradrenaline. Specifically, HFD suppressed the normal pattern of periodic [Ca2+ ]c oscillations in isolated hepatocytes and disrupted the propagation of intralobular [Ca2+ ]c waves in the intact perfused liver. Short-term HFD inhibited noradrenaline-induced inositol 1,4,5-trisphosphate generation, but did not change basal endoplasmic reticulum Ca2+ load or plasma membrane Ca2+ fluxes. We propose that impaired Ca2+ signalling plays a key role in the earliest phases of the etiology of NAFLD, and is responsible for many of the ensuing metabolic and related dysfunctional outcomes at the cellular and whole tissue level.

Fmr1 exon 14 skipping in late embryonic development of the rat forebrain.

BACKGROUND: Fragile X syndrome, the major cause of inherited intellectual disability among men, is due to deficiency of the synaptic functional regulator FMR1 protein (FMRP), encoded by the FMRP translational regulator 1 (FMR1) gene. FMR1 alternative splicing produces distinct transcripts that may consequently impact FMRP functional roles. In transcripts without exon 14 the translational reading frame is shifted. For deepening current knowledge of the differential expression of Fmr1 exon 14 along the rat nervous system development, we conducted a descriptive study employing quantitative RT-PCR and BLAST of RNA-Seq datasets. RESULTS: We observed in the rat forebrain progressive decline of total Fmr1 mRNA from E11 to P112 albeit an elevation on P3; and exon-14 skipping in E17-E20 with downregulation of the resulting mRNA. We tested if the reduced detection of messages without exon 14 could be explained by nonsense-mediated mRNA decay (NMD) vulnerability, but knocking down UPF1, a major component of this pathway, did not increase their quantities. Conversely, it significantly decreased FMR1 mRNA having exon 13 joined with either exon 14 or exon 15 site A. CONCLUSIONS: The forebrain in the third embryonic week of the rat development is a period with significant skipping of Fmr1 exon 14. This alternative splicing event chronologically precedes a reduction of total Fmr1 mRNA, suggesting that it may be part of combinatorial mechanisms downregulating the gene's expression in the late embryonic period. The decay of FMR1 mRNA without exon 14 should be mediated by a pathway different from NMD. Finally, we provide evidence of FMR1 mRNA stabilization by UPF1, likely depending on FMRP.

Effects of Magnetite Nanoparticles and Static Magnetic Field on Neural Differentiation of Pluripotent Stem Cells.

Neurodevelopmental processes of pluripotent cells, such as proliferation and differentiation, are influenced by external natural forces. Despite the presence of biogenic magnetite nanoparticles in the central nervous system and constant exposure to the Earth's magnetic fields and other sources, there is scant knowledge regarding the role of electromagnetic stimuli in neurogenesis. Moreover, emerging applications of electrical and magnetic stimulation to treat neurological disorders emphasize the relevance of understanding the impact and mechanisms behind these stimuli. Here, the effects of magnetic nanoparticles (MNPs) in polymeric coatings and the static external magnetic field (EMF) were investigated on neural induction of murine embryonic stem cells (mESCs) and human induced pluripotent stem cells (hiPSCs). The results show that the presence of 0.5% MNPs in collagen-based coatings facilitates the migration and neuronal maturation of mESCs and hiPSCs in vitro. Furthermore, the application of 0.4 Tesla EMF perpendicularly to the cell culture plane, discernibly stimulates proliferation and guide fate decisions of the pluripotent stem cells, depending on the origin of stem cells and their developmental stage. Mechanistic analysis reveals that modulation of ionic homeostasis and the expression of proteins involved in cytostructural, liposomal and cell cycle checkpoint functions provide a principal underpinning for the impact of electromagnetic stimuli on neural lineage specification and proliferation. These findings not only explore the potential of the magnetic stimuli as neural differentiation and function modulator but also highlight the risks that immoderate magnetic stimulation may affect more susceptible neurons, such as dopaminergic neurons.

Receptor-specific Ca2+ oscillation patterns mediated by differential regulation of P2Y purinergic receptors in rat hepatocytes.

Extracellular agonists linked to inositol-1,4,5-trisphosphate (IP3) formation elicit cytosolic Ca2+ oscillations in many cell types, but despite a common signaling pathway, distinct agonist-specific Ca2+ spike patterns are observed. Using qPCR, we show that rat hepatocytes express multiple purinergic P2Y and P2X receptors (R). ADP acting through P2Y1R elicits narrow Ca2+ oscillations, whereas UTP acting through P2Y2R elicits broad Ca2+ oscillations, with composite patterns observed for ATP. P2XRs do not play a role at physiological agonist levels. The discrete Ca2+ signatures reflect differential effects of protein kinase C (PKC), which selectively modifies the falling phase of the Ca2+ spikes. Negative feedback by PKC limits the duration of P2Y1R-induced Ca2+ spikes in a manner that requires extracellular Ca2+. By contrast, P2Y2R is resistant to PKC negative feedback. Thus, the PKC leg of the bifurcated IP3 signaling pathway shapes unique Ca2+ oscillation patterns that allows for distinct cellular responses to different agonists.

A Tale of two receptors.

Calcium (Ca2+) oscillations in hepatocytes have a wide dynamic range. In particular, recent experimental evidence shows that agonist stimulation of the P2Y family of receptors leads to qualitatively diverse Ca2+ oscillations. We present a new model of Ca2+ oscillations in hepatocytes based on these experiments to investigate the mechanisms controlling P2Y-activated Ca2+ oscillations. The model accounts for Ca2+ regulation of the IP3 receptor (IP3R), the positive feedback from Ca2+ on phospholipase C (PLC) and the P2Y receptor phosphorylation by protein kinase C (PKC). Furthermore, PKC is shown to control multiple cellular substrates. Utilising the model, we suggest the activity and intensity of PLC and PKC necessary to explain the qualitatively diverse Ca2+ oscillations in response to P2Y receptor activation.

Pathophysiology in the comorbidity of Bipolar Disorder and Alzheimer's Disease: pharmacological and stem cell approaches.

Neuropsychiatric disorders involve various pathological mechanisms, resulting in neurodegeneration and brain atrophy. Neurodevelopmental processes have shown to be critical for the progression of those disorders, which are based on genetic and epigenetic mechanisms as well as on extrinsic factors. We review here common mechanisms underlying the comorbidity of Bipolar Disorders and Alzheimer's Disease, such as aberrant neurogenesis and neurotoxicity, reporting current therapeutic approaches. The understanding of these mechanisms precedes stem cell-based strategies as a new therapeutic possibility for treatment and prevention of Bipolar and Alzheimer's Disease progression. Taking into account the difficulty of studying the molecular basis of disease progression directly in patients, we also discuss the importance of stem cells for effective drug screening, modeling and treating psychiatric diseases, once in vitro differentiation of patient-induced pluripotent stem cells provides relevant information about embryonic origins, intracellular pathways and molecular mechanisms.